Examples of Custom-order Application list

Application Packages

Reprogramming characterization (phase contrast)

Background
In human iPS cell reprogramming, improvement of reprogramming efficiency is a very important issue. A number of critical conditions, including optimizing transformation conditions and the types of compounds applied, have been studied and improved. But even under the best conditions, there remains a subset of colonies that appear during iPS cell reprogramming that are not completely reprogrammed. In order to distinguish iPS cell colonies from non-iPS cell colonies, various laborious and expensive evaluation steps are employed. These steps include manual determination of colony number via visual observation or by fixing and dyeing cells for manual quantification. Determining good quality iPS cell colonies in live cultures early in the reprogramming process will shorten reprogramming time and reduce the cost of reagents to be used. Therefore, a rapid and noninvasive colony determination method is required.
Solution
Determine iPSC colony and non-iPSC colony to evaluate reprogramming efficiency using morphological features of colony outline.
Usage
Evaluation of reprogramming efficiency.

Sequential iPS cell counting (phase contrast)

Background
Cell growth of human iPS cells is an important criterion for determining healthy cell conditions. Because hPSCs are usually dissociated into cell clumps of 50-100 cells for passaging, separate counting of cells is time-consuming, labor-intensive, and wasteful of the available cells.
Solution
Count cell number on images from single cell to iPSC colony without dissociating cell aggregation and losing cells.
Usage
Cell counting.

iPS cell colony distribution map (phase contrast)

Background
During passaging, human iPS cell colonies are dissociated into clumps or single cells and then seeded into new culture vessels. Once plated, iPS cells immediately gather together to form colonies. Non-uniform distribution of cells/colonies affects cell growth and pluripotency, resulting in variation of cell conditions. Skillful and careful handling is required for uniform distribution of colonies in culture vessels.
Solution
Numeralize the cell density of culture the dish surface and create density distribution map, recognizing cell attached regions.
Usage
Evaluation of culture irregularity.

iPS cell colony characterization (phase contrast)

Background
Because it is difficult to maintain human pluripotent stem cells (hPSCs) in a healthy state, hPSCs should be routinely characterized using several established examination techniques. These techniques should be employed during cell culture expansion for research and/or therapeutic purposes. hPSC colony morphology is typically considered an important criterion. However, evaluation by manually observing colony morphology is not quantitative.
Solution
Automatically distinguish undifferentiated state of cells inside and around hPSC colony, recognizing iPS cell colony regions.
Usage
Evaluation of differentiation status.

Sequential merged colony characterization (phase contrast)

Background
During passaging, human iPS cell colonies are dissociated into clumps or single cells and then seeded into new culture vessels. Immediately after seeding these cells gather together to form colonies. These colonies grow larger over time, and as they grow they may begin to merge with each other. This merging sometimes leads to differentiation. Therefore careful monitoring of colony-to-colony contacts is critical.
Solution
Determine the degree of fusion between iPSC colonies with tracking of individual iPSC colonies from the start.
Usage
Evaluation of colony fusion degree.

Neuronal maturation classification (fluorescence, phase contrast)

Background
Methods for establishing disease specific iPS cells are being developed. If patient derived iPS cells can be established, it is expected that these cells can be used to produce large quantities of cells/tissues of the central nervous system, which are currently difficult to procure from a living human body and use for drug screening. However, to be used for drug discovery applications, highly efficient methods for neuronal differentiation of iPS cells are required.
Solution
Quantify induction efficiency and induction rate when iPS cells are induced to differentiate into neurons from cell body area, length of neurites and the number of connecting points between neurite and cell body.
Usage
Evaluation of differentiation status.

Measurement Applications

Sequential total colony coverage ratio (phase contrast)

Background
Adherent cultures should be passaged when they are in the log growth phase, before they reach confluence when the culture vessel is fully covered with cells. The optimal time to subculture cells is usually when culture vessel coverage reaches 60-80% of total culture vessel area (a.k.a. subconfluent). Even though this process is a routine part of the cell culture process, it is still difficult to determine when the cells should be passaged. This is because, in most cases, total cell coverage is judged subjectively using manual microscopic inspection without consistent and objective quantification. It is critical that cells are passaged prior to confluence if proper differentiation outcomes from hPSCs are to be consistently achieved.
Solution
Identify hPSC colonies based on image criteria and measure the total colony-occupied area on the culture vessel. This application can monitor sequential total colony coverage ratio.
Usage
Determination of the cell passage timing; In-process evaluation of cell culture condition; In-process quality evaluation of hPSC.

Sequential colony size (phase contrast)

Background
Measuring the increase in colony size over time is an essential part of monitoring hPSC health and phenotype. Colony size increases directly reflect the nature of growth and proliferation of cells in the colony. Whether seeded as single cells or clumps of cells, undifferentiated iPS/ES cells form a distinctive colony. These colonies tend to be very heterogeneous in their sizes. Assays that track colony size provide a quantitative measure of hPSC phenotypic integrity.
Solution
Identify individual hPSC colonies based on image criteria and track the size/area of each colony over time.
Usage
In-process quality evaluation of hPSC; In-process phenotypic evaluation of the hPSC colony; Identification of transformed cells.

Colony compactness classification (phase contrast)

Background
Cell density within hPSC colonies can vary significantly during culture. In general, within a few days after passage, the morphologies of the hPSCs are comparatively flatter and larger than those of typical undifferentiated hPSCs, and spaces between the cells are recognizable in small colonies. In contrast, colonies at several days after passage come to have tightly packed, rounded cells without minimal intercellular spacing. The cells within these colonies have large nuclei with notable nucleoli. hPSC colony density and compactness measurements reflect the phenotype and so-called "maturity" of the hPSC colony and can be used to determine suitable timing for subculture of mature colonies.
Solution
Distinguish the high density of cells in the hPSC colony based on the image analysis and judge how "mature" the colony is.
Usage
Determination of the isolation timing of the hPSC colony; Determination of the cell passage timing; In-process evaluation of cell culture condition.

Differentiated area classification (phase contrast)

Background
Maintaining the undifferentiated state of hPSCs can be challenging. Culture of hPSCs requires careful handling according to a strict regimen of growth factors, specialized media and plate coating, and often cumbersome cell handling. Even with careful adherence to this strict culture regimen, a certain percentage of hPSC cells can spontaneously differentiate. And, prolonged culture of hPSCs can lead to genomic/epigenomic abnormalities, further complicating hPSC culture by resulting in uncontrollable growth of undifferentiated cells and/or undesired differentiation. Identifying differentiated regions of the hPSC colony early and with accuracy is critical to successful culture. This has traditionally been done by immunofluorescence staining using specific antibodies against undifferentiated and differentiated cell surface markers. This process takes time and requires fixation and immuno-labeling of cells. Post-fixation it is not possible to use them for downstream processes such as expansion, differentiation, preservation, etc. Thus identifying differentiated regions in live hPSC colonies using label-free, non-invasive methods is essential for researchers to utilize hPSCs for downstream processes.
Solution
Identify differentiated regions in or around colonies.
Usage
Identification of differentiated cells; Go/no-go monitoring based on hPSC differentiation.

Undifferentiated area classification (phase contrast)

Background
Maintaining the undifferentiated state of hPSCs can be challenging. Culture of hPSCs requires careful handling according to a strict regimen of growth factors, specialized media and plate coating, and often cumbersome cell handling. Even with careful adherence to this strict culture regimen, a certain percentage of hPSC cells can spontaneously differentiate. And, prolonged culture of hPSCs can lead to genomic/epigenomic abnormalities, further complicating hPSC culture by resulting in uncontrollable growth of undifferentiated cells and/or undesired differentiation. Distinguishing undifferentiated regions from differentiated regions of the hPSC colony early and with accuracy is critical to successful culture. This has traditionally been done by immunofluorescence staining using specific antibodies against undifferentiated and differentiated cell surface markers. This process takes time and requires fixation and immuno-labeling of cells. Post-fixation it is not possible to use them for downstream processes such as expansion, differentiation, preservation, etc. Thus identifying undifferentiated regions in live hPSC colonies using label-free, non-invasive methods is essential for researchers to utilize hPSCs for downstream processes.
Solution
Identify undifferentiated regions within the hPSC colony.
Usage
In-process quality evaluation of the hPSC undifferentiated status.

Induced colony counting (phase contrast)

Background
Improving reprogramming efficiency is crucial to obtain appropriate iPS cell lines to pass through the various quality evaluations required for defining the pluripotency. Research into methods for enhancing reprogramming efficiency is ongoing. Through changing various conditions, including using different combinations of genes and adding small and large molecules, reprogramming efficiency can be improved significantly. Determination of reprogramming efficiency is usually done by manual microscopic observation or counting of fixed and stained colonies. Post fixation it is not possible to use cells for downstream processes such as expansion, differentiation, preservation, etc. Thus determining the reprogramming efficiency by non-invasive methods is essential for researchers to utilize them for further downstream processes.
Solution
Identify human iPSC colonies by non-invasive phase contrast imaging and count the number of iPSC colonies.
Usage
Measurement of the reprogramming efficiency.

Induced colony classification (phase contrast)

Background
Establishing fully reprogrammed iPS cells requires multiple validations that are laborious and costly. In addition, not all of the colonies that appear during reprogramming consist of fully reprogrammed iPS cells. Therefore identification and isolation of true iPS cell colonies early in the reprogramming process can greatly reduce culturing time, laborious examinations and the cost of reagents.
Solution
Determine iPSC or non-iPSC colonies based on their shape, morphology, growth rate etc based on image analysis.
Usage
Measurement of the reprogramming efficiency.

Reprogramming efficiency (phase contrast)

Background
Improving the reprogramming efficiency is crucial to obtain enough iPS cell lines that can pass through various quality evaluations required for defining the pluripotency. Researchers have been studying to enhance the reprogramming efficiency by changing various conditions such as different combinations of genes and adding small and large molecules. It is often that the determination of the efficiency is based on manual microscopic observation or counting of colonies with invasive fixation followed by staining. Thus determining the reprogramming efficiency by the non-invasive and quantitative method is essential for researchers to utilize them for further downstream processes.
Solution
Identify human iPSC colonies by the non-invasive phase contrast imaging. Evaluate the colony quality based imaging criteria such as shape, morphology, growth rate. Measure the reprogramming efficiency by counting the number of true iPSC colonies.
Usage
Measurement of the reprogramming efficiency.

Sequential cell counting (phase contrast)

Background
Mesenchymal stem cells (MSCs) are multipotent stem cells that have been isolated from adult tissues, including bone marrow, umbilical cord, and adipose tissue. However, proliferation of MSCs is highly inconsistent and declines with an increasing number of passages. Also, primary cultured cells from older donors tend not to expand well. Therefore it is important to evaluate the growth activity of MSCs over time to assess their health and quality throughout the culture process. Current methods rely on manual microscopic inspection without consistent and objective quantification. Having an objective, quantitative method for measuring MSC health in culture is critical for efficient large-scale production of MSCs.
Solution
Sequentially measure cell numbers from non-invasive phase contrast images. The cells can be used for subsequent processes.
Usage
In-process evaluation of cell culture condition, Evaluation of culturing cells based on the growth.

Cell adhesion ratio (phase contrast)

Background
The ratio of adherent vs. floating cells in culture post-passage can serve as an indicator of how supportive to cell health the culture conditions are. Even after successful passaging, if optimal cell culture conditions are not maintained, cells loose their ability to adhere to the culture substrate as they undergo cell death. Current methods for determining the cell adhesion ratio use cell fixation and staining with dye, or manual microscopic inspection. Having an objective, quantitative, label-free method for measuring the cell adhesion ratio in culture is critical for efficient monitoring of culture conditions in hPSC cultures.
Solution
Identify and measure the subpopulation of adherent/suspended cells from non-invasive phase contrast images.
Usage
In-process evaluation of cell culture condition. Evaluation of culturing cells based on the subpopulation of adherent cells.

Sequential cell coverage area ratio (phase contrast)

Background
It is considered desirable that cells should be subcultured when they are 70-80% confluent (subconfluent). It is critical that cells are passaged prior to confluence to keep phenotype pf MSCs. Current methods rely on manual microscopic inspection without consistent and objective quantification. Having an objective, quantitative method for measuring the coverage area ratio in culture is critical for efficient stable production of MSCs.
Solution
Identify MSC cells from non-invasive phase contrast images and calculate the cell coverage area of culture vessel.
Usage
Decision of the timing of passage. In-process evaluation of cell culture condition, evaluation of culturing cells based on the cell growth.

Sequential cell density distribution (phase contrast)

Background
MSCs show promise as materials for biological studies and clinical applications in regenerative medicine. However proliferation activity of MSC is easily affected by culture conditions and requires careful attention. Improper cell culturing skills can alter and spoil the quality and character of MSCs. For example, uniform cell distribution in culture vessel is one of the crucial indexes for evaluating the cell culturing process. Although cell density in a culture vessel is commonly measured by an operator's visual assessment, using an objective, non-biased quantitative method to measure cell density and its distribution ensures MSC culture conditions are optimal and eliminates operator error.
Solution
Measure the cell density and distribution from phase contrast images and visualize these features in heat map style. Uniformness of cell distribution and resultant growth ability can be quantified.
Usage
Recording cell culture processes and evaluation of operator's skill.

Sequential cell density (phase contrast)

Background
MSCs show promise as materials for biological studies and clinical applications in regenerative medicine. Proliferation activity of MSCs is highly affected by culture conditions and micro-environment, both of which require careful care and attention. Cell density during culture is a crucial parameter used to evaluate the microenvironment during the cell culture process, and for determining the timing for subculture. Using an objective, non-biased quantitative method to measure cell density ensures MSC culture conditions are optimal and provides guidance for proper passage timing.
Solution
Measure the cell density by identification of individual MSC cell from phase contrast images.
Usage
In-process evaluation of cell culture condition, evaluation of culturing cells based on the cell density measurement.

Sequential cell size (phase contrast)

Background
MSCs have a finite proliferative potential that decreases with each passage. It is known that highly proliferative MSCs have a relatively small surface area while senescent cells become larger during the senescence process. Current cell size assessments rely on visual inspection by technicians. A more robust, quantitative method is required for efficient determination of MSC size.
Solution
Identify individual cells and measure their size from phase contrast images which are taken under conventional cell culturing conditions. Individual cell size is an important index of condition and state.
Usage
In-process evaluation of cell culture condition, evaluation of culturing cells based on the cell area.

Morphological classification (phase contrast)

Background
In recent years, it has been accepted that conventional MSC surface markers are not sufficient to identify the quality of MSC. New methods that rely on morphology are open to a new possibility of identifying actively proliferating MSCs. MSCs have a finite proliferative potential that decreases with each passage. It is known that highly proliferative MSCs are comparatively small and rounded but become larger and elongated during their senescence process. Current morphology assessments rely on visual inspection by technicians. A more robust, quantitative method is required for efficient culture of MSCs.
Solution
Quantify cell morphological features using morphological parameters such as the cell area, cell roundness, peripheral length and number of cells from non-invasive phase contrast images.
Usage
In-process evaluation of cell culture condition, evaluation of culturing cells based on the cell morphological features.

Cell migration (phase contrast)

Coming soon.


Sequential single neuron classification (fluorescence)

Background
Neurons show a very distinctive morphology that is different from many other somatic cells. Neurons have protrusions extending from the cell body that output signals to other cells, as well as axons and dendritic bifurcations that diverge like branches of a tree and receive signals from other cells. These morphological features are thought to be important for measuring the nature and health status of neurons, but because of their very complex shape they are difficult to evaluate quantitatively.
Solution
Count cell body number and neurite number by time-lapse single cell tracking analysis.
Usage
Drug discovery assay targeting nerve cells, single cell tracking analysis aiming at differentiation evaluation of noninvasive iPSC-derived neurons.

Sequential single neuron classification (phase contrast)

Background
Neurons show a very distinctive morphology that is different from many other somatic cells. Neurons have protrusions extending from the cell body that output signals to other cells, as well as axons and dendritic bifurcations that diverge like branches of a tree and receive signals from other cells. These morphological features are thought to be important for measuring the nature and health status of neurons, but because of their very complex shape they are difficult to evaluate quantitatively.
Solution
Count cell body number and neurite number by time-lapse single cell tracking analysis.
Usage
Drug discovery assay targeting nerve cells, single cell tracking analysis aiming at differentiation evaluation of noninvasive iPSC-derived neurons.

Cell counting (phase contrast)

Background
Neurons show a very distinctive morphology that is different from many other somatic cells. Neurons have protrusions extending from the cell body that output signals to other cells, as well as axons and dendritic bifurcations that diverge like branches of a tree and receive signals from other cells. These morphological features are thought to be important for measuring the nature and health status of neurons, but because of their very complex shape they are difficult to evaluate quantitatively.
Solution
Count cell body number by end point analysis.
Usage
Drug discovery assay targeting nerve cells, endpoint analysis aiming at differentiation evaluation of noninvasive iPSC-derived neurons.

Cell body size (phase contrast)

Background
Neurons show a very distinctive morphology that is different from many other somatic cells. Neurons have protrusions extending from the cell body that output signals to other cells, as well as axons and dendritic bifurcations that diverge like branches of a tree and receive signals from other cells. These morphological features are thought to be important for measuring the nature and health status of neurons, but because of their very complex shape they are difficult to evaluate quantitatively.
Solution
Assess cell body area from outline of cell body by end point analysis.
Usage
Drug discovery assay targeting nerve cells, endpoint analysis aiming at differentiation evaluation of noninvasive iPSC-derived neurons.

Node classification (fluorescence)

Background
Neurons show a very distinctive morphology that is different from many other somatic cells. Neurons have protrusions extending from the cell body that output signals to other cells, as well as axons and dendritic bifurcations that diverge like branches of a tree and receive signals from other cells. These morphological features are thought to be important for measuring the nature and health status of neurons, but because of their very complex shape they are difficult to evaluate quantitatively.
Solution
Count number of nodes per neuron by end point analysis.
Usage
Drug discovery assay targeting nerve cells, endpoint analysis aiming at differentiation evaluation of noninvasive iPSC-derived neurons.

Branch classification (fluorescence)

Background
Neurons show a very distinctive morphology that is different from many other somatic cells. Neurons have protrusions extending from the cell body that output signals to other cells, as well as axons and dendritic bifurcations that diverge like branches of a tree and receive signals from other cells. These morphological features are thought to be important for measuring the nature and health status of neurons, but because of their very complex shape they are difficult to evaluate quantitatively.
Solution
Count branching points per neuron by end point analysis.
Usage
Drug discovery assay targeting nerve cells, endpoint analysis aiming at differentiation evaluation of noninvasive iPSC-derived neurons.

Neurite length (fluorescence)

Background
Axons and dendrites that extend from the cell bodies of nerve cells mediate signal transduction between nerves and play an essential role in the construction of neural networks. In patients with neurodegenerative diseases such as Alzheimer's, atrophy of axons and dendrites, as well as cell death caused by dendritic processes, is observed. These parameters can be quantified and serve as important measures of neuronal cell health.
Solution
Measure neurite length by end point analysis.
Usage
Drug discovery assay targeting nerve cells, endpoint analysis aiming at differentiation evaluation of noninvasive iPSC-derived neurons.

Neurite length (phase contrast)

Background
Axons and dendrites that extend from the cell bodies of nerve cells mediate signal transduction between nerves and play an essential role in the construction of neural networks. In patients with neurodegenerative diseases such as Alzheimer's, atrophy of axons and dendrites, as well as cell death caused by dendritic processes, is observed. These parameters can be quantified and serve as important measures of neuronal cell health.
Solution
Measure neurite length by end point analysis.
Usage
Drug discovery assay targeting nerve cells, endpoint analysis aiming at differentiation evaluation of noninvasive iPSC-derived neurons.